U.S. Pat. No. 6,594,011 issued Jul. 15, 2003, the entirety of which is incorporated by reference herein for all purposes, discloses an imaging apparatus and method for real time imaging ellipsometry for high throughput sensing of binding events useful in molecular interaction analysis including biotech applications. The apparatus and method disclosed employ the immobilization of an array of binding or capture molecules (“ligands”) on a planar surface of a transparent substrate and the use of a beam of polarized light directed at the underside of the surface in a manner to achieve total internal reflection (TIR) and generate an evanescent field in the plane of the ligands. The ligands are exposed to a biological sample and analytes in the biological sample bind to different patterns of the immobilized ligands in a manner to change the polarization at locations in the array at which binding occurs. An image of the array is compared with a stored image of the initial light polarization shifts to determine the location and magnitude of binding events within the array, thus identifying and quantitating the analytes present in the biological sample.
The apparatus for implementing the foregoing technique typically employs a prism or gratings to achieve the requisite TIR generated evanescent field, the prism being the more practical implementation.
TIR imaging ellipsometry works well for fields of view up to 1-2 cm2, which permits real time imaging of tens of thousands of binding events simultaneously. However, there is a need to be able to image or scan areas which are much larger, such as 128 mm×86 mm (e.g., the area of a 384 well or a 96 multiwell plate) to permit lower costs per test and for multiple tests per patient for large numbers of patients simultaneously which is increasingly a requirement for more clinical diagnostics and personalized medicine. Obviating the need for a single large prism simplifies both the instrument and disposable multiwell plate.
Co-pending U.S. application Ser. No. 11/696,369, filed Apr. 4, 2007, the entirety of which is incorporated by reference herein for all purposes, discloses a multiwell plate in which arrays of ligands are printed on planar side walls of the liquid reservoirs, or wells. The plate is fabricated with transparent material, such as glass or plastic, and a beam of light is directed upwards from the bottom of the plate into the separation between adjacent wells. The direction of the beam is chosen to achieve total internal reflection (TIR) at a well side wall in a manner to generate an evanescent field in the plane of an array of ligands on the interior face of that well. The reflected light from the side wall carries binding information between analytes in a biological sample in the selected well and different patterns of capture molecules in the array immobilized on the addressed side wall.